ABSTRACT Genome instability has long been implicated as a main causal factor in cancer, neurodegeneration and aging. Ribonucleotide (rNMP) represents the most abundant non-canonical nucleotide in genomic DNA and is a major source of genome instability. Understanding how rNMPs are incorporated into DNA is important for defining fundamental mechanisms of genome instability and disease pathogenesis. rNMP incorporation by DNA polymerases is a major source of rNMP contamination in DNA, however the efficiency and frequency of rNMP insertion are unknown for a novel DNA polymerase/primase (PrimPol). A knowledge gap exists in the understanding of the contribution of PrimPol to rNMP contamination in DNA and in the functions of its protein-interaction partners. PrimPol is a newly discovered, versatile human translesion synthesis (TLS) DNA polymerase/primase that is fundamentally distinct from many other human TLS pols. This is because in addition to DNA lesion bypass, PrimPol can perform de novo DNA/RNA synthesis and origin-independent re-priming. The long-term goal of the project is to elucidate the mechanisms of genetic instability, determine the mechanisms of key enzymes involved, and developing novel strategies to reduce genome instability and its pathogenic effects. The objective of this application is to determine the contribution of PrimPol to rNMP incorporation in DNA relative to several other important TLS pols, and to elucidate the regulatory functions of its protein interaction partner, replication protein A (RPA). The central hypothesis is that PrimPol contributes genome instability via rNMP incorporation and RPA regulates this activity. This hypothesis will be tested with two aims. Aim 1, to quantify the efficiency, frequency and sequence specificity of PrimPol-catalyzed rNMP incorporation using steady-state, pre-steady-state kinetic analyses, LC-MS based oligonucleotide sequencing and computer simulations. Several cancer-related variants will be characterized for its replication fidelity, rNMP incorporation frequency and catalytic competency. Aim 2, to elucidate the role of RPA in modulating PrimPol-catalyzed nucleotide incorporation using steady-state kinetic analysis. A novel competitive assay will be developed to assess the preference of PrimPol for its DNA polymerase and primase activities. The proposed work is significant because it will advance the understanding of novel mechanisms of genomic instability and disease pathogenesis, which will inform the development of new therapeutics. This proposal is innovative because it will, for the first time, quantitatively define the contribution of PrimPol and its variants to rNMP contamination in DNA, and determine the biological functions of RPA-PrimPol interactions, which will advance our current understanding of the biological function of PrimPol and its role in genomic instability. The proposed research program will offer meaningful biomedical training opportunities for students and greatly enhance the overall biomedical research environment at Central Michigan University.